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1 Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium
2 Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Science, Ghent University, 9000 Gent, Belgium
Correspondence
Hans J. Nauwynck
hans.nauwynck{at}ugent.be
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ABSTRACT |
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Supplementary material is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Phagocytosis is an active and highly regulated process involving specific cell surface receptors and signalling cascades mediated by Rho GTPases (Hall & Nobes, 2000; Conner & Schmid, 2003). Pinocytosis can occur through several pathways. The cargo and its receptor determine which pinocytic pathway will be used (Conner & Schmid, 2003). A first possible route for pinocytosis is ‘macropinocytosis’ (Swanson & Watts, 1995). Other pathways are named after the main structural compound involved: ‘clathrin-mediated endocytosis’ (Brodsky et al., 2001) and ‘caveolae-mediated endocytosis’ (Pelkmans & Helenius, 2002). Finally, there is a group called ‘clathrin- and caveolae-independent pathways’. They are differentiated based on their dependency on cholesterol and/or association with lipid rafts, dynamin and Rho GTPases (Nichols & Lippincott-Schwartz, 2001; Conner & Schmid, 2003). Viruses are specialized in misusing these endocytic pathways to gain entry into the cell (Sieczkarski & Whittaker, 2002a; Pelkmans & Helenius, 2003).
Recently, we have demonstrated that feline infectious peritonitis virus (FIPV), a devastating coronavirus in cats, enters its host cell, the monocyte, via endocytosis (Van Hamme et al., 2007). The pathway used is unknown. Transmissible gastroenteritis virus (TGEV), canine coronavirus and human coronavirus 229E (HCoV 229E) are coronaviruses that – like FIPV – belong to phylogenetic group 1. They have all been shown to enter cells via endocytosis (Hansen et al., 1998; Savarino et al., 2003; Nomura et al., 2004). The pathways used for internalization have not been studied, except for HCoV 229E which enters human fibroblast cells through the caveolae-mediated pathway (Nomura et al., 2004). Severe acute respiratory syndrome (SARS), an emerging disease in man, is caused by a coronavirus belonging to phylogenetic group 2. Due to its threat, many studies have been done to understand better how the virus interacts with its host and host cells (Kim et al., 2006). It was shown that SARS virus enters HepG2 cells expressing ACE2 via pH-dependent endocytosis through clathrin-coated vesicles (Yang et al., 2004; Inoue et al., 2007). More recently, SARS virus was shown to enter Vero E6 cells through receptor-mediated, clathrin- and caveolae-independent endocytosis, likely involving lipid rafts (Wang et al., 2008). The entry of murine coronavirus, also belonging to group 2, is extensively studied. Although entry via non-endosomal routes has been suggested, recent publications assign a major role for cholesterol-dependent endocytosis, possibly through clathrin-coated pits, in murine cells (Thorp & Gallagher, 2004; Choi et al., 2005; Eifart et al., 2007).
In this study, the mechanism of internalization of FIPV in monocytes was determined through chemical inhibition of internalization, transduction of monocytes with constructs to induce expression of dominant-negative (DN) proteins that hinder some pathways and co-localization studies. Our results indicate that FIPV is internalized through a clathrin- and caveolae-independent pathway that strongly depends on dynamin and is slightly cholesterol depletion sensitive.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and seeded onto glass coverslips in medium [RPMI 1640 enriched with 10 % fetal bovine serum, 0.3 mg glutamine ml–1, 1 mM sodium pyruvate and 1 % non-essential amino-acids (100x; Gibco-Invitrogen)]. Cells consisted of 86±7 % monocytes. FIPV serotype II strain 79-1146 was kindly provided by Dr Egberink (Faculty of Veterinary Medicine, Utrecht University, The Netherlands) and passaged in a feline cell line (CrFK) (McKeirnan et al., 1981). Batches of culture fluids were purified by ultracentrifugation and purified virus was biotinylated as described previously (Van Hamme et al., 2007). Purified viral-particle suspension (200 µg) was applied to 104.4 monocytes throughout the experiments. This amount of suspension contains 5.0x1010 virus particles of which 1.9x105 particles are infectious (Van Hamme et al., 2007). This suspension was used exclusively in entry assays, not in the infection and co-localization studies.
Entry inhibition assay.
Monocytes were washed extensively at 68 h post-seeding and pre-incubated at 37 °C for 30 min with one of the following compounds dissolved in RPMI 1640 (Gibco-Invitrogen) (all products were purchased from Sigma-Aldrich; unless stated otherwise): 0.1 µM wortmannin, 20 µM latrunculin B (ICN Biochemicals), 2 µM chlorpromazine, 500 µM amantadine, 50 µg nystatin ml–1, 10 mM methyl-β-cyclodextrin, 50 µg genistein ml–1, 40 and 80 µM dynamin inhibitory peptide (dip) (batch 5; Tocris Cookson) or 0.74 nM Clostridium difficile toxin B. Working concentrations were optimized qualitatively in internalization assays with control ligands, while insuring that the viability of the cells was always over 99 %. After pre-treatment, monocytes were inoculated with biotinylated FIPV in the presence of the compound used for pre-treatment. Cells and virus were incubated at 37 °C for 1 h. Then, cells were briefly washed with ice-cold RPMI 1640 and fixed with formaldehyde (1 %). Bound particles were stained with streptavidin–Texas red (Molecular Probes) and after permeabilization with Triton X-100 (0.1 %) internalized particles were stained with streptavidin–fluorescein isothiocyanate (FITC) (Molecular Probes). After mounting of the coverslips, internalization was quantified per cell as the ratio of internalized virus particles to the total number of particles. At least 10 cells were analysed and all particles over the entire volume of the cell were monitored. The procedure described above, from seeding of the cells up to the analysis, was performed three times.
To assess the effectiveness of all agents, internalization of biotinylated transferrin (Sigma-Aldrich), FITC-labelled albumin and fluorescent 1 µm FluoSpheres (Molecular Probes) was studied in the presence of appropriate inhibitors. After fixation and permeabilization, biotinylated transferrin was visualized with streptavidin–FITC and in the cells incubated with fluorescent beads, cortical actin was stained using falloidin–Texas red (Molecular Probes). Coverslips were mounted on microscope slides and analysed by confocal microscopy. Cells were scored as internalizing cells if all control ligands were internalized. For each experiment, 50–100 cells were analysed and experiments were performed three times.
Plasmid constructions and production of lentiviral supernatants.
Transfer vectors were prepared by deletion of green fluorescent protein (GFP) from the TRIP
U3-CMV-GFP-WPRE vector (=TRIPU3-CMV-WPRE). The enhanced GFP (EGFP) tagged DN eps15 construct, named DIII and the EGFP tagged control construct D32, kindly provided by Dr Benmerah (Benmerah et al., 1998), were excised from pEGFP-C2 and cloned into TRIPU3-CMV-WPRE. EGFP tagged wild-type (WT) and DN caveolin-1, kindly provided by Dr Helenius (Kurzchalia et al., 1992; Pelkmans et al., 2001), and WT and DN dynamin 2(aa), gifted by Dr McNiven (Cao et al., 1998, 2000) were also cloned into TRIPU3-CMV-WPRE. Biological activity of the constructs in the original plasmids and after transfer into pTRIPU3-CMV-WPRE was tested in CrFK cells.
pMD.G and p8.91 were used as envelope and packaging plasmids, respectively, as described previously (Stove et al., 2005). At 70 % confluency, 293FT cells (Invitrogen) were co-transfected with 1.66 µg packaging plasmids, 3.33 µg envelope plasmids and 3.33 µg transfer plasmids using a calcium phosphate transfection kit (Invitrogen). Lentiviral supernatants were harvested after 40 h (Stove et al., 2005).
Inhibition of virus internalization pathways through lentiviral gene transfer.
At 3 h post-seeding, cells were washed and medium was replaced by lentiviral supernatants. At 24 h post-seeding, cells were washed and fresh medium was added. Internalization assays were performed at 68 h post-seeding. After washing the cells, biotinylated FIPV was added and incubated with the cells for 1 h at 37 °C. Then, cells were briefly washed with ice-cold RPMI 1640 and fixed with formaldehyde (1 %). Bound particles were stained with streptavidin–Alexa Fluor 350 (Molecular Probes) and after permeabilization with Triton X-100 (0.1 %), internalized particles were stained with streptavidin–Texas red. Coverslips were mounted and the level of internalization was quantified as described above.
For the controls, transduced cells were incubated with biotinylated transferrin or biotinylated cholera toxin B (Sigma-Aldrich). After fixation and permeabilization, ligands were visualized with streptavidin–Texas red. Coverslips were mounted onto microscope slides and analysed by confocal microscopy. Cells were scored as internalizing cells if all control ligands were internalized.
Co-localization with clathrin.
At 68 h post-seeding, cells were washed with RPMI 1640 (37 °C) and incubated with non-biotinylated FIPV at an m.o.i. of 5 for 0, 5, 15 and 45 min at 37 °C. Then, cells were washed with RPMI 1640 and fixed. Cells were washed again, first with RPMI 1640 followed by Tris-buffered saline (TBS; 20 mM Tris/HCl, 150 mM NaCl, pH 7.5) with 4.5 % sucrose and 2 % inactivated goat serum (TBS-GS) and permeabilized with methanol for 30 s at –20 °C (Racoosin & Swanson, 1994). Clathrin was stained with anti-clathrin heavy-chain IgM antibodies (ICN Biochemicals), diluted 1 : 50 in PBS supplemented with 0.3 % gelatin (PBS-G) (Van de Walle et al., 2001; Misinzo et al., 2005). Afterwards, cells were washed in TBS-GS and incubated for 1 h at 37 °C with biotin-labelled goat anti-mouse IgM antibodies (Santa Cruz Biotechnology) diluted 1 : 100 in PBS-G. Then, cells were washed and incubated with streptavidin–Texas red (Molecular Probes) diluted 1 : 50 in PBS-G. After washing, FIPV was stained with anti-FIPV–FITC [Veterinary Medical Research Development (VMRD), Pullman, USA]. Coverslips were mounted onto microscope slides and analysed by confocal microscopy.
Co-localization with caveolin-1.
At 68 h post-seeding, cells were washed with RPMI 1640 (37 °C) and incubated with non-biotinylated FIPV at an m.o.i. of 5 for 0, 5, 15 and 45 min at 37 °C. Then, cells were washed with RPMI 1640 and fixed. Cells were permeabilized with Triton X-100 (0.1 %) and washed with PBS. Caveolin-1 was stained with polyclonal rabbit anti-caveolin-1 antibodies (Abcam), diluted 1 : 200 in PBS, by incubation for 1 h at 37 °C. Afterwards, cells were washed and incubated for 1 h at 37 °C with Texas red-labelled goat anti-rabbit antibodies (Molecular Probes) diluted 1 : 100 in PBS. After washing, FIPV was stained with anti-FIPV–FITC. Coverslips were mounted onto microscope slides and analysed by confocal microscopy.
Infection inhibition assay.
At 56 h post-seeding, cells were washed with RPMI 1640 and pre-incubated for 30 min at 37 °C with inhibitory compounds (wortmannin, latrunculin B, chlorpromazine, amantadine, nystatin, genistein, dip and toxin B) dissolved in RPMI 1640. Then, cells were inoculated with non-biotinylated FIPV (m.o.i. of 1 except for dip and the corresponding control: m.o.i. of 0.1 of FIPV grown in serum-free medium) in the presence of the inhibitors in RPMI and incubated at 37 °C. After 1 h the inoculum was washed off and cells were treated with a trypsin/EDTA (0.25 %/0.02 % in RPMI) solution for 5 min at 37 °C to remove bound, non-internalized virus particles from the plasma membrane (for evidence of removal of non-internalized virus particles by this method, see Supplementary Material, including Supplementary Fig. S1, available in JGV Online). Thereafter, cells were extensively washed with RPMI 1640 and medium was added. After 11 h of incubation at 37 °C, cells were washed, fixed and permeabilized. Permeabilization was followed by 1 h of incubation at 37 °C with anti-FIPV–FITC and 10 min with Hoechst 33342 (Molecular Probes). Coverslips were mounted onto microscope slides and analysed by confocal microscopy. Cells with cytoplasmic expression of viral proteins were scored as infected cells. All cells on the coverslips were evaluated.
Microscopy and statistics.
Internalization and infection assays were analysed by a DM IRB inverted microscope (Leica Microsystems). Images of internalization assays were obtained with a Leica TCS SP2 laser scanning spectral confocal system linked to a DM IRB inverted microscope (Leica). Argon and He/Ne lasers were used for exciting FITC and Texas red fluorochromes, respectively. Leica confocal software was used for image acquisition.
Triplicate assays were performed and compared using the Mann–Whitney U test from the SPSS software package (version 12.0, SPSS). P
0.05 were considered significantly different.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Araki et al., 1996). The influence of these compounds on the internalization of FIPV in monocytes was studied. Wortmannin and latrunculin B reduced the uptake of control ligands (fluorescent beads) significantly to 36.6±4.0 and 17.2±3.8 %, respectively, relative to the untreated controls. The internalization of FIPV was unaffected: 104.7±9.9 % for wortmannin and 100.1±9.0 % for latrunculin B relative to the untreated controls (Fig. 1).
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; Wang et al., 1993). Transferrin uptake occurs constitutively via clathrin-mediated endocytosis, and was therefore used as a control (Harding et al., 1983). After treatment of monocytes with chlorpromazine and amantadine, transferrin uptake was reduced significantly to 28.9±0.4 and 13.0±15.4 %, respectively, of the control. The internalization of FIPV, however, remained at the same level (94.0±5.0 and 99.3±9.2 % of the control for chlorpromazine and amantadine, respectively), despite the presence of inhibitors (Fig. 1
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Secondly, monocytes were transduced with a control construct (D32) or with DN eps15 (DIII). eps15 protein is crucial for clathrin-mediated endocytosis (Benmerah et al., 1998). To assess the effectiveness of the constructs, internalization of transferrin was studied in transduced cells: in cells with D32, the uptake was 103.3±21.7 % relative to the uptake in untransduced control cells; in DIII transduced cells, the uptake was 47.9±8.5 % relative to the control. Thus, the uptake of transferrin was reduced significantly relative to the uptake in D32 transduced cells. The internalization of FIPV showed no reduction: in cells with D32, the uptake was 98.3±0.7 % relative to the uptake in untransduced control cells; in DIII transduced cells, the uptake was 90.3±10.6 % relative to the control. Thus, transduction with DN eps15 did not influence the cell's ability to internalize FIPV (Fig. 2a and b).
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). Taken together, the experiments clearly show that clathrin is not involved in the internalization of FIPV in monocytes.
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; Simons & Toomre, 2000). Therefore, sterol-binding drugs, like nystatin and methyl-β-cyclodextrin, disrupt the caveolar function (Rothberg et al., 1992). Genistein blocks caveolae-mediated internalization through inhibition of protein tyrosine kinases. The effect of these drugs on FIPV internalization was investigated. Albumin is internalized through caveolae and was used as a control ligand (Schnitzer et al., 1994). Nystatin and methyl-β-cyclodextrin reduced the uptake of albumin to 54.9±14.5 and 22.2±4.4 %, respectively, of the control. The uptake of FIPV was reduced slightly to 85.0±1.4 and 87.4±6.1 %, respectively, of the control (Fig. 1). Genistein reduced internalization of albumin significantly to 32.6±5.3 % of the control. However, internalization of FIPV was unaffected (107.1±10.7 % of the control) (Fig. 1). Thus, despite the slight effect of sterol-binding drugs, caveolae are probably not involved in FIPV entry as it is not affected by genistein.
Another experiment was performed to confirm that caveolae do not play a role in the internalization of FIPV. The most important protein compounds of the caveolae are caveolins. The presence of DN caveolin-1 directly inhibits the endocytic process through caveolae (Pelkmans et al., 2001; Pelkmans & Helenius, 2002). Monocytes were transduced with either WT or DN caveolin-1. To confirm the effectiveness, the uptake of Cholera toxin B was studied in transduced cells (Montesano et al., 1982; Parton et al., 1994): in cells with WT caveolin-1, the uptake was 105.1±3.2 % relative to the uptake in untransduced control cells and in cells with DN caveolin-1, the uptake was 34.8±7.8 % relative to the uptake in untransduced control cells. For FIPV, no reduction was observed: in cells with WT caveolin-1, FIPV uptake was 102.1±3.4 % and in cells with DN caveolin-1, FIPV uptake remained at 100.1±7.1 % relative to the untransduced control (Fig. 2c and d). These results indicate that caveolin-1 is not involved in FIPV entry.
Further, most FIPV protein clusters (93.1±4.1 %) did not co-localize with caveolin-1 between 0 and 45 min after the start of virus uptake (Fig. 4). Only occasionally, particles were found in the proximity of caveolin-1. A co-localization study between caveolin-1 and an irrelevant protein (transferrin) showed that the observed level of co-localization does not exceed the expected level for coincidental co-localization (data not shown).
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FIPV entry depends on dynamin
Dynamin is a GTPase that is involved in many internalization pathways: phagocytosis, clathrin-mediated internalization, caveolae-mediated internalization and clathrin- and caveolae-independent pathways (Gold et al., 1999; Hinshaw, 2000; Mayor & Pagano, 2007). Dynamin is thought to pinch off formed vesicles from the plasma membrane (Hinshaw, 2000). Cells were treated with dip and analysed for internalization of the control ligand transferrin and FIPV (Grabs et al., 1997). Dip (40 µM) reduced transferrin uptake significantly to 27.4±8.4 % relative to the uptake in untreated cells. For FIPV, a strong, significant reduction was observed to 40.1±9.6 and 20.3±1.1 % of the uptake in control cells for 40 and 80 µM, respectively, of dynamin inhibitor (Fig. 1). To confirm these results, the uptake of FIPV was studied in cells transduced with WT and DN dynamin 2(aa) (Cao et al., 1998, 2000). The effectiveness of transduction was studied by monitoring the uptake of transferrin in transduced cells: in cells with WT dynamin 2(aa), the uptake was 86.5±13.4 % relative to the uptake in untransduced control cells and in cells with DN dynamin 2(aa), the uptake was 39.0±12.2 % relative to the uptake in untransduced control cells. Internalization of FIPV was reduced significantly in cells expressing DN dynamin 2(aa), compared with cells expressing WT dynamin 2(aa): in cells with WT dynamin 2(aa), FIPV uptake was 105.6±6.3 % and in cells with DN dynamin 2(aa), FIPV uptake was 58.7±3.9 % relative to the uptake in untransduced control cells (Fig. 2e and f). Clearly, dynamin plays an important role in the internalization of FIPV in monocytes.
Rho GTPases are not involved in FIPV entry
Several independent internalization pathways have been characterized by their dependency on Rho GTPases (Mayor & Pagano, 2007). Rho GTPases are a subfamily of the Ras superfamily of small GTPases. They play an important role in regulating the actin cytoskeleton and in a broad range of aspects of endocytic traffic (Hall, 1998; Ellis & Mellor, 2000). Rho GTPases are involved in phagocytosis and macropinocytosis and also in clathrin- and caveolae-mediated internalization pathways (Ellis & Mellor, 2000; Grimmer et al., 2002). To determine whether they are involved in the internalization of FIPV in monocytes, cells were treated with Clostridium difficile toxin B, a general Rho GTPase inhibitor (Just et al., 1995). The activity of the inhibitor was confirmed by its effect on the internalization of fluorescent beads: a significant reduction to 10.0±2.2 % of the uptake in untreated cells (Fig. 1). The internalization of FIPV was not significantly affected: internalization remained at the level of 89.2±11.1 % of the control. These results were confirmed by less general Rho GTPase inhibitors like Rac1 inhibitor (Calbiochem), secramine A [inhibits Cdc42 activation (Pelish et al., 2006)] and Y-27632 (selective inhibitor of the Rho-associated protein kinase, ROCK; Sigma-Aldrich) (data not shown).
The effect of entry inhibitors on FIPV infection
After establishing the effect of inhibitors on the uptake of virus, the effect on infection was studied. Therefore, cells were inoculated and incubated with FIPV in absence and in presence of entry inhibitors. Bound, non-internalized virions were removed from the cell surface by trypsin wash. Viral replication was stopped after 1 cycle of replication, i.e. at 12 h post-inoculation. Fig. 5 shows that the inhibitors that did not significantly influence FIPV entry, had no effect on infection rates. The actual influence of the inhibitors on percentages of infected cells were from 2.1 to 1.9 % for wortmannin, 1.9 to 1.7 % for latrunculin B, 2.1 to 2.1 % for chlorpromazine, 1.9 to 2.3 % for amantadine, 1.9 to 1.9 % for genistein and 1.9 to 2.4 % for toxin B. However, inhibition of FIPV internalization by dip is reflected in significantly reduced infection (45.7±10.3 and 23.0±9.5 % of infection of control cells for 40 and 80 µM of dynamin inhibitor, respectively, or reductions from 0.48 to 0.22 and 0.32 to 0.065 % infected cells). Nystatin treatment during entry led to a reduction to 61.7±5.2 % of infection of control cells or absolutely from 1.9 to 1.2 % infected cells.
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DISCUSSION |
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). In the present study, the mechanism of endocytosis involved in FIPV entry was determined. Multiple strategies were combined: internalization pathways were blocked by use of chemical inhibitors, and expression of DN mutants and co-localization assays were performed. FIPV is not phagocytosed nor macropinocytosed as the inhibitors wortmannin and latrunculin B did not affect the entry and Rho GTPases were not involved in the internalization. Independency from clathrin was proven based on the facts that inhibitions with chlorpromazine and amantadine were ineffective and that expression of DN eps15 had no effect on FIPV internalization. FIPV and clathrin did not co-localize either. Further, it was shown that caveolae are not involved in FIPV internalization as genistein and expression of DN caveolin-1 had no effect on FIPV internalization, and FIPV and caveolin-1 did not co-localize inside the cell. In conclusion, FIPV enters monocytes through a clathrin- and caveolae-independent pathway. This pathway is further characterized by its dependency on dynamin and independency from Rho GTPases.
Further, the sterol-binding drugs nystatin and methyl-β-cyclodextrin slightly affected FIPV internalization. Cholesterol depletion affects internalization pathways associated with lipid rafts. The association with rafts can be crucial for initiating and proceeding signalling cascades necessary for internalization. However, requirement for cholesterol is not necessarily related to lipid rafts. For example, cholesterol depletion disturbs clathrin-mediated internalization, which is not associated with lipid rafts (Subtil et al., 1999; Nichols & Lippincott-Schwartz, 2001). As the observed decrease in internalization of FIPV is much smaller than the decrease in internalization of the control ligand and as the observed reduction due to methyl-β-cyclodextrin is smaller than those described in literature (ranging from 30 to >90 % for different pathways) (Rodal et al., 1999; Subtil et al., 1999; Grimmer et al., 2002; Sanchez-San Martin et al., 2004; Barrias et al., 2007), it seems likely that the pathway is not dependent on cholesterol. Possibly, a fraction of the virus-binding receptor is present in lipid rafts. Disturbance of rafts could then cause a decrease in receptor availability. Reduced virus binding subsequently leads to a drop in the number of internalized virions. Further research is needed to confirm the effect of sterol-binding drugs and to enlighten the underlying cause.
To our knowledge, no identical physiological internalization pathway has been described. However, the characteristics of the internalization of the β-chain of interleukin 2 receptors (IL-2R-β) are very similar to those of FIPV internalization (Lamaze et al., 2001). Like FIPV, IL-2R-β is internalized via a clathrin- and caveolae-independent pathway and is dependent on dynamin and sensitive to cholesterol depletion. The sensitivity to cholesterol depletion is caused by raft-association of the receptor. The only difference with the uptake of FIPV, is the dependency on Rho GTPases. The internalization of the common cytokine receptor 
(c) is categorized in the same group as the IL-2R-β-pathway (Kirkham & Parton, 2005; Sauvonnet et al., 2005). The c receptor is a subunit shared by several cytokine receptors, namely the IL-2, -4, -7, -9, -15 and -21 receptors (Schluns & Lefrancois, 2003). When expressed by itself, the c receptor is rapidly and efficiently endocytosed (Morelon & Dautry-Varsat, 1998). Clathrin- and caveolae-independent pathways have also been linked to the entry of many viruses (Marsh & Helenius, 1989; Sieczkarski & Whittaker, 2002a; Sanchez-San Martin et al., 2004). Like FIPV, SARS virus is internalized through a clathrin- and caveolae-independent pathway that is sensitive to treatment with methyl-β-cyclodextrin (Wang et al., 2008). The involvement of dynamin and Rho GTPases has however not been studied. Rotavirus cell entry resembles FIPV entry as it is also internalized via a clathrin- and caveolae-independent pathway that depends on dynamin, but the pathway is highly cholesterol depletion sensitive. The role of Rho GTPases in this endocytic process has not been studied either. In addition, there are similarities between the entry pathways used by influenza virus and FIPV. Influenza virus enters HeLa cells via a clathrin- and caveolae-independent pathway that is not associated with lipid rafts. It is not known whether this pathway depends on dynamin (Sieczkarski & Whittaker, 2002b). Influenza virus entry in Mv-1 lung cells has been shown to depend on dynamin but further characterization has not been performed (Roy et al., 2000). As the route of entry depends on the host cell type, it is impossible to predict whether influenza virus entry into HeLa cells will also depend on dynamin. Thus, for both FIPV and influenza virus, the internalization pathways need further characterization to compare them.
HCoV 229E belongs to the same phylogenetic group as FIPV and is the only member of the group of which the internalization process has been characterized. It was shown that HCoV 229E binds to its receptor, human aminopeptidase N (APN), in rafts and enters human fibroblasts through caveolae (Nomura et al., 2004). Human APN has been reported to be a component of rafts in various cell types (Danielsen, 1995; Santos et al., 2000; Riemann et al., 2001; Nomura et al., 2004). For TGEV, that is endocytosed after binding to porcine APN, it has been suggested that clathrin might be involved in the internalization (Hansen et al., 1998). For type II FIPV, feline APN is the receptor for entry in cell lines. Whether this is also a receptor in primary target cells is not known. Even if APN would be the internalizing receptor, no conclusions can be made based on the entry mechanisms of HCoV and TGEV as these coronaviruses already use two different mechanisms of internalization through APN.
Despite the low number of infected cells, which is an inevitable obstacle for FIPV research in target cells, infection assays in the presence of entry inhibitors qualitatively confirmed the results from the internalization assays and show that virions infect their host cell via the described pathway. Inhibition of dynamin function and nystatin treatment reduced infection. The reduction in infection caused by nystatin is larger than expected based on the reduction of internalization. Possibly, cholesterol binding by nystatin affects post-entry events, e.g. intracellular transport, despite the trypsin wash.
In this study, the mechanism of internalization for FIPV 79-1146 in monocytes was determined. The pathway is clathrin- and caveolae-independent, strongly depends on dynamin and is slightly cholesterol depletion sensitive. The identified pathway proceeds similarly, though not exactly like IL-2R-β endocytosis, and SARS- and rotavirus entry. Gaining insights in the initial virus–cell interactions is valuable in the search for an effective treatment or prevention of FIP. Obviously, the pathway should be further characterized and its components should be identified in order to obtain well-defined targets for antiviral therapy.
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ACKNOWLEDGEMENTS |
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Received 26 February 2008;
accepted 30 April 2008.
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